PROJECT SUMMARY Cell surface receptors enable cells to detect and respond to signals in their external environment. G protein-coupled receptors (GPCRs) are both the largest class of cell surface receptors in humans, and the receptor family most targeted by drugs. It has long been thought that GPCR signaling was limited to the cell surface. However, this paradigm has shifted with the recent discovery of GPCR signaling from internalized endosomes. Endosomal pH is thought to be a key factor determining the duration of endomembrane signaling: as endosomes mature their internal pH declines from 7.0 to 5.0. This pH change has the potential to modulate GPCR-drug interactions, regulate receptor conformation, and prioritize receptor-effector interactions. Similar pH effects caused by pathologies such as cancer, asthma, and cystic fibrosis have the potential to modulate GPCR-drug interactions at the cell surface. Studying the relationship between pH and GPCR function enables us to identify receptors (in)capable of endomembrane signaling, and (in)sensitive to disease-related acidosis. However, the effect of pH on GPCR signaling remains poorly understood. The goal of this proposal is to fill this gap in knowledge. Using our integrated computational and experimental screening platform, we aim to interrogate the pH sensitivities of a minimum of five receptors per project year. By the end of the 5-year project period we anticipate having evaluated a minimum of 25 GPCRs from a prioritized list of receptors with known structures, drug interactions, and clinical applications. Computationally, we would use our structure- based informatics and virtual ligand screening software, known as pHinder, to identify pH-sensitive residue hot spots, charge networks between receptors and ligands, and pH-selective drug compounds. Experimentally, we would confirm our computational predictions, interrogate GPCR-drug interactions, and screen against >2,000 drug-like compounds using our novel yeast-based pH-screening platform. We expect that the outcomes of our structure-based calculations, in silico and cell-based drug screens, and yeast-based pH-screening assays will open new and unexpected avenues in GPCR pharmacology. Specifically, we anticipate that these efforts will illuminate the molecular basis GPCR pH sensing, identify GPCR-drug interactions that are (dys)regulated by endosomal and pathological pH changes, and spur the development of drugs for selectively modulating receptors at neutral and/or acidic pH.